Present day solid-phase methods for the synthesis of peptides or proteins are largely based on the original methodology developed by Merrifield, employing a functionalized cross-linked styrene/divinylbenzene copolymer, the cross-linked copolymer having been formed by the polymerization of styrene monomer to which has been added a few per cent (typically about 2%) of divinylbenzene. This copolymer generally provided in the form of beads or particles, often with a dominant particle size of 20-80 .mu.m. The functionalization originally preferred by Merrifield [see e.g. J. Am. Chem. Soc. 85, 2149 (1963)] was a functionalization of the aromatic rings of the copolymer with chloromethyl groups, introduced via reaction of the solid copolymer with SnCl.sub.4 /chloromethyl methyl ether, although a number of other functionalities, including aminomethyl, .alpha.-aminobenzyl and .alpha.-amino-4-methylbenzyl, have subsequently been employed. Regardless of its nature, the purpose of the functionality is normally to form an anchoring linkage between the copolymer solid support and the C-terminal of the first amino acid which it is desired to couple to the solid support. More recent refinements of the Merrifield methodology have included the further introduction, between a functionality (e.g. one of the above-mentioned functionalities) on the polystyrene chains and the C-terminal of the first amino acid which is to be coupled, of a bifunctional "spacer" or "handle" group whose reactivity is tailored inter alia to meet desired requirements with respect both to the coupling of the first amino acid to the solid support and/or to the ease with which the completed, synthesized peptide or protein chain is cleaved from the solid support. Examples of such spacer groups include the phenylacetamidomethyl (Pam) and the p-alkoxybenzyl ester systems. A recent review dealing with the development of solid-phase peptide synthesis methodology since its introduction by Merrifield is given by Barany et al. [Int. J. Peptide Protein Res. 30, 705-739 (1987)].
Recent advances in biotechnology, particularly in the area of recombinant DNA, have produced a unique situation: the availability and rapid accumulation of many new protein sequences with undefined or unknown function and/or unknown biological activity. Detailed structural analysis by site-directed mutagenesis or similar molecular engineering has provided a useful approach concerning the roles of amino acid residues in active sites of proteins.
However, specific information concerning biologically active functional subunits containing ca. 5-40 amino acid residues is preferably obtained through chemical synthesis. Current solid-phase technology is quite sufficient to yield such peptides reliably and in high purity, but the conventional method of solid-phase peptide synthesis via a "linear" mode of approach produces only one peptide per synthesis.
A method employing a "simultaneous" or "parallel" mode of approach to the synthesis of peptides is thus desirable, thereby facilitating the production of a large number of peptides which can, for example, be used to define and map the functional entities of proteins.
A basic feature of the solid-phase technique of peptide synthesis is that in each elongation of the peptide chain with a further amino acid, all treatment steps are repetitive of the previous cycle with the possible exception of the amino acid coupling step itself, in which a further amino acid that may or may not be identical with that coupled in the preceding cycle is coupled to the peptide chain. Thus, a parallel, substantially simultaneous synthesis of more than one peptide can be achieved by performing in parallel the repetitive steps, such as deprotection, neutralization, and washing, which are common to the parallel syntheses. The major technical difficulty is the attainment of compartmentalization of each amino acid coupling step so that cross-contamination will not occur.
Two different methods have recently been proposed for the substantially simultaneous synthesis of a number of peptides:
The first of these methods [Geysen et al., Proc. Natl. Acad. Sci. USA. 81, 3998-4002 (1984) and 82, 178-82 (1985)] was devised for rapid screening of peptide epitopes via ELISA (Enzyme Linked Immunosorbent Assay) in 96-microtiter wells. It utilizes acrylic acid-grafted polyethylene rod-and-96-microtiter wells to immobilize growing peptide chains and to perform the compartmentalized synthesis. However, while highly effective, the method is not applicable on a preparative scale, i.e. to the ,preparation of milligram quantities. The second method [Houghten, Proc. Natl. Acad. Sci. USA. 82, 5131-35 (1985)] utilizes a "tea bag" containing the traditionally used polymer beads to compartmentalize the synthesis, portions of peptidylresin beads being kept apart in sealed bags of fine-mesh polypropylene net. The latter method is relevant to the preparation of milligram quantities.
The obvious advantages of a method permitting the parallel and substantially simultaneous synthesis of a multitude of peptides are the attendant saving in time and the redundancy of the repetitive labour involved in accomplishing the synthesis of each peptide individually.